A new guideline for molecular testing to select lung cancer patients for treatment with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors has been jointly developed by the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.¹ The guideline, recently published in Archives of Pathology and Laboratory Medicine, provides evidence-based recommendations on which patients and samples should be tested and how testing should be performed.

The recommendations are based on a review of literature published between January 2004 and February 2012. Co-chairs and writing and advisory panels consisted of members of all three societies. Expert opinion was also solicited from representatives of FDA, ASCO, the National Cancer Institute, and the National Comprehensive Cancer Network.

The recommendations are framed as answers to 14 questions on individual topics, as summarized below. “Recommendations” are supported by grade A or B evidence. “Suggestions” are supported by grade C evidence. “Expert consensus opinion” was used to designate guidance for which grade C or better evidence is lacking.

When Should Molecular Testing of Lung Cancer Be Performed?

Which patients should be tested for EGFR mutations and ALK rearrangements?

It is recommended that EGFR and ALK molecular testing be used to select patients for EGFR- and ALK-targeted tyrosine kinase inhibitor treatment and that patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics (Recommendation).

For resection specimens, testing is recommended for adenocarcinomas and mixed lung cancers with an adenocarcinoma component regardless of histologic grade; for fully excised specimens, testing is not recommended in lung cancers that lack any adenocarcinoma component (Recommendation). For more-limited specimens (biopsy, cytology), testing may be performed in cases showing squamous or small cell histology (Recommendation).

Primary tumors or metastatic lesions are equally suitable for testing (Recommendation). Separate primary lung tumors in an individual patient may be tested separately (Expert consensus opinion).

When should a specimen be tested?

EGFR mutation (Recommendation) and ALK rearrangement (Suggestion) testing should be ordered at time of diagnosis of patients with advanced-stage disease (stage IV) or at the time of recurrence or progression in patients initially presenting with lower-stage disease. EGFR and ALK testing at diagnosis of stage I, II, or III disease is encouraged, but the decision should be made locally by each laboratory in collaboration with its oncology team (Expert consensus opinions). Tissue should be prioritized for EGFR and ALK testing (Recommendation).

How rapidly should test results be available?

EGFR and ALK results should be available within 2 weeks (10 working days) of receipt of the specimen in the testing laboratory. Laboratories with longer average turnaround times need to make a more rapid test available in cases of clinical urgency. Laboratory departments should establish processes to ensure that specimens with a final histopathologic diagnosis are sent to outside molecular pathology laboratories within 3 working days or to intramural laboratories within 24 hours (Expert consensus opinions).

How Should EGFR Testing Be Performed?

How should specimens be processed for EGFR testing?

Pathologists should use formalin-fixed, paraffin-embedded specimens or fresh, frozen, or alcohol-fixed specimens for polymerase chain reaction (PCR)-based EGFR tests. Other tissue treatments should be avoided. Cytologic samples are suitable for EGFR and ALK testing, with cell blocks preferred over smear preparations (Expert consensus opinions).

What are the specimen requirements for EGFR testing?

Pathologists should determine the adequacy of specimens by assessing cancer cell content and DNA quantity and quality. Each laboratory should establish the minimum proportion and number of cancer cells needed for mutation detection during validation. A pathologist should assess the tumor content of each specimen and perform (or guide a trained technologist in performing) microdissection for tumor cell enrichment as needed (Expert consensus opinions).

How should EGFR testing be performed?

Laboratories may use any validated EGFR testing method with sufficient performance characteristics (Recommendation). Test methods that detect mutations in specimens with at least 50% cancer cell content should be used, although laboratories are strongly encouraged to use tests able to detect mutations in specimens with as little as 10% cancer cells (Expert consensus opinion). Mutation testing should be able to detect all mutations that have been reported with a frequency of at least 1% in lung adenocarcinomas (Expert consensus opinion).

Neither immunohistochemistry for total EGFR nor EGFR copy number analysis (eg, fluorescence [FISH] or chromogenic [CISH] in situ hybridization) is recommended for selection of EGFR tyrosine kinase inhibitor therapy (Recommendations).

What is the role of KRAS analysis in selecting patients for EGFR tyrosine kinase inhibitor therapy?

KRAS mutation testing is not recommended as a sole determinant of EGFR tyrosine kinase inhibitor therapy (Recommendation).

What additional considerations are important in the setting of secondary or acquired EGFR tyrosine kinase inhibitor resistance?

Tests should be able to detect the secondary EGFR T790M mutation in as few as 5% of cells (Recommendation).

How Should ALK Testing Be Performed?

What methods should be used for ALK testing?

Labs should use an ALK FISH assay with dual-labeled break-apart probes for selecting patients for ALK tyrosine kinase inhibitor therapy. If it is carefully validated, ALK immunohistochemistry may be considered for screening to select specimens for ALK FISH testing (Recommendation). Reverse transcriptase PCR is not currently recommended for selecting patients for ALK tyrosine kinase inhibitor treatment (Recommendation).

A pathologist should be involved in selecting sections for ALK FISH testing by assessing tumor architecture, cytology, and specimen quality, and a pathologist should participate in interpretation of ALK FISH slides by performing the analysis directly or reviewing the interpretation of cytogeneticists or technologists with specialized training in solid tumor FISH analysis (Expert consensus opinions).

Testing for secondary ALK mutations associated with acquired resistance to ALK inhibitors is not currently required for clinical management (Expert consensus opinion).

Should Other Genes Be Routinely Tested?

Are other molecular markers suitable for testing in lung cancer?

Testing for EGFR should have priority over testing for other molecular markers in lung adenocarcinoma (Recommendation), and testing for ALK, after testing for EGFR, should take priority over testing for proposed markers (Suggestion). Published evidence is currently insufficient to support development of testing guidelines for other proposed markers.

How Should Molecular Testing Be Implemented and Operationalized?

Must all adenocarcinomas be tested for both EGFR and ALK?

Simultaneous testing is difficult to implement due to high costs and labor requirements. Laboratories may implement step-wise testing algorithms to enhance efficiency of molecular testing, provided the overall turnaround time requirements are met (Expert consensus opinion).

How should EGFR and ALK test results be reported?

Reports should include a results and interpretation section that can be readily understood by oncologists and nonspecialist pathologists (Expert consensus opinion).

How should EGFR and ALK testing be validated?

EGFR and ALK testing validation should follow the same guidelines as for other molecular diagnostics ad FISH tests (Expert consensus opinion).

How should quality assurance be maintained?

Laboratories should follow quality control and quality assurance policies for EGFR and ALK testing similar to those for other clinical laboratory assays. Laboratories performing such testing for tyrosine kinase inhibitor therapy should enroll in proficiency testing, if available. ■

Reference

1. Lindeman NI, Cagle PT, Beasley MB, et al: Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors. Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Arch Pathol Lab Med. April 3, 2013 (early release online).